CN109863997B - Tissue culture method of Mongolian mulberry seedlings - Google Patents

Tissue culture method of Mongolian mulberry seedlings Download PDF

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CN109863997B
CN109863997B CN201910236128.0A CN201910236128A CN109863997B CN 109863997 B CN109863997 B CN 109863997B CN 201910236128 A CN201910236128 A CN 201910236128A CN 109863997 B CN109863997 B CN 109863997B
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subculture
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CN109863997A (en
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赵泉胜
铁英
白玉娥
彭鹏
邰海欧
李红颖
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Inner Mongolia Hesheng Ecological Technology Research Institute Co ltd
Mengshu Ecological Construction Group Co ltd
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Inner Mongolia Hesheng Ecological Technology Research Institute Co ltd
Mengshu Ecological Construction Group Co ltd
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Abstract

The invention provides a tissue culture method of a Mongolian mulberry seedling, which takes a dormant bud of the Mongolian mulberry as an explant material and MS as a basic culture medium, and 6-BA, 2,4-D, IBA and GA with different concentrations are added into the culture medium3And PVP-40 and the like, and sequentially carrying out subculture, multiplication culture and rooting culture on the mulberries explant to obtain a complete plant. The method can obtain a large amount of Mongolian mulberry regenerated seedlings, and solves the problems of variation and slow growth caused by seed propagation and the problem of disease and insect pest propagation caused by a cuttage mode. The method can obviously improve the breeding coefficient of the Mongolian mulberry and shorten the breeding period. In the examples of the present invention, the growth phase was grown at a rate of 9.3 times, and the rooting rate was 100%.

Description

Tissue culture method of Mongolian mulberry seedlings
Technical Field
The invention relates to the technical field of forest tree breeding, in particular to a tissue culture method of a Mongolian mulberry seedling.
Background
Mongolian mulberry (Morus mongolica), also known as "Yansang", is an ideal ecological and economic tree species, and plays a very positive role in soil and water conservation and ecological restoration in northern areas. Mainly distributed in the mountainous region of Daqingshan, the loess hilly region of the south of the Yin mountain, the mountainous region of the south of Daxing' anling, and the natural protection region of the Daqinggou of the branch of the Cladong.
The Mongolian mulberry is a cross-pollinated plant, the seedling cultured by sowing is easy to generate variation, the excellent characters of a female parent cannot be maintained, the seedling period of the seedling grows slowly, the seedling period is long, and the propagation coefficient is low. Although the cutting propagation can maintain the excellent characters of the female parent, the diseases and the insect pests are easy to accumulate, the quantity requirement on the female tree and the destructiveness on the tree type are high, and the popularization and the planting of the excellent seedlings of the Mongolian mulberry are greatly restricted.
The seedling produced by plant tissue culture can preserve the character of mother tree, and its production is not affected by time, season and environment, and can implement mass propagation of seedling in short time. In recent years, with the development of technology, the cost of tissue culture is reduced, the number of breeding is increased continuously, the market price of the good Mongolian mulberry seedlings can be effectively lowered, and the promotion effect on the breeding and popularization of new varieties is greatly promoted. However, there is currently no corresponding tissue culture method for the Morus mongolicus seedlings.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a tissue culture method of Mongolian mulberry seedlings.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the invention relates to a tissue culture method of Mongolian mulberry seedlings, which comprises the following steps:
(1) taking a mulberry leaf explant, and carrying out sterilization treatment to obtain a sterile explant;
(2) inoculating the sterile explant into an induction culture medium for induction culture to obtain an induced seedling;
(3) inoculating the induced seedling into a subculture medium for subculture to obtain a subculture seedling;
(4) inoculating the subculture seedling into a proliferation culture medium for proliferation culture to obtain cluster buds;
(5) and inoculating the cluster buds into a rooting culture medium for rooting culture to obtain rooted seedlings.
Preferably, in step (1), the explant is a dormant bud of Morus mongolica.
Preferably, in the step (1), the sterilization treatment is to wash the explant with sterile water after soaking the explant with an alcohol solution and a mercuric chloride solution in sequence.
Preferably, in the step (1), the volume fraction of the alcohol solution is 75%, and the mass concentration of the mercuric chloride solution is 0.1%.
Preferably, in the step (2), the induction medium is based on an MS medium and is obtained by adding 6-BA, IBA, PVP-40, sucrose and agar, wherein the concentration of 6-BA in the induction medium is 1.0mg/L, the concentration of IBA is 0.5mg/L, the concentration of PVP-40 is 500mg/L, the concentration of sucrose is 20g/L, the concentration of agar is 5.5g/L, and the pH value of the induction medium is 5.8-6.0.
Preferably, in the step (2), the inducing culture is to inoculate the sterile explant into an inducing culture medium, and culture is carried out for more than 20 days under the conditions that the temperature is 25 +/-2 ℃, the illumination intensity is 1500-2000 Lx and the illumination time is 10-14 h/day until the sterile explant grows to about 3cm, so as to obtain the induced seedling.
Preferably, in step (3), the subculture medium is based on 1/2MS medium, and IBA and GA are added3Sucrose and agar, the concentration of IBA in the subculture medium is 0.5mg/L, and GA3The concentration of (3) is 3mg/L, the concentration of sucrose is 20g/L, the concentration of agar is 5.5g/L, and the pH value of the subculture medium is 5.8-6.0.
Preferably, in the step (3), the subculture is to inoculate the induced seedling into a subculture medium, and culture is carried out for more than 30 days under the conditions that the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lx and the illumination time is 15-17 h/day until the induced seedling roots and grows to 5-7 cm, so as to obtain the subculture seedling.
Preferably, in the step (4), the enrichment medium is based on an MS medium and is obtained by adding 6-BA, 2,4-D, sucrose and agar, wherein the concentration of 6-BA in the enrichment medium is 1.5mg/L, the concentration of 2,4-D is 0.3mg/L, the concentration of sucrose is 30g/L, the concentration of agar is 5.5g/L, and the pH value of the enrichment medium is 5.8-6.0.
Preferably, in the step (4), the propagation culture is to cut a small segment with an axillary bud from the subculture seedling, inoculate the small segment in a propagation culture medium, and culture for more than 30 days under the conditions that the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lx, and the illumination time is 15-17 h/day, until the small segment of the subculture seedling grows to 5-7 cm, so as to obtain the cluster bud.
Preferably, in the step (5), the rooting medium is obtained by taking 1/2MS medium as a basis and adding IBA, sucrose and agar, wherein the concentration of IBA in the rooting medium is 0.3mg/L, the concentration of sucrose is 20g/L, the concentration of agar is 5.5g/L, and the pH value of the rooting medium is 5.8-6.0.
Preferably, in the step (5), the rooting culture is to inoculate the cluster buds into a rooting culture medium, and the cluster buds are subjected to rooting culture for 20-25 days under the conditions of the temperature of 25 +/-2 ℃, the illumination intensity of 2500-3000 Lx and the illumination time of 15-17 h/d to obtain rooted seedlings.
Preferably, after the step (5) is finished, the method further comprises the step (6): and (4) cleaning the rooted seedlings, and culturing in a matrix for 15-20 days to obtain transplanted seedlings.
Preferably, the volume ratio of the grass carbon to the vermiculite to the perlite in the matrix is 3: 2: 1.
the invention has the beneficial effects that:
the invention provides a tissue culture method of a Mongolian mulberry seedling, which takes a dormant bud of the Mongolian mulberry as an explant material and MS as a basic culture medium, and 6-BA, 2,4-D, IBA and GA with different concentrations are added into the culture medium3And PVP-40 and the like, and sequentially carrying out induction culture, subculture, multiplication culture and rooting culture on the mulberry explants to obtain complete plants. The method can obtain a large amount of Mongolian mulberry regenerated seedlings, and solves the problems of variation and slow growth caused by seed propagation and the problem of disease and insect pest propagation caused by a cuttage mode. The method can obviously improve the breeding coefficient of the Mongolian mulberry and shorten the breeding period. In the examples of the present invention, the growth phase was grown at a rate of 9.3 times, and the rooting rate was 100%.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
The embodiment of the invention relates to a tissue culture method of Mongolian mulberry seedlings, which comprises the following steps:
(1) and (3) taking the mulberry leaf explant for sterilization treatment to obtain the sterile explant.
Wherein, the explant is a segment of plant organ or tissue as an isolated culture material, and has the characteristics of vigorous metabolism and strong regeneration capacity. In one embodiment of the invention, the explant is a dormant bud of Morus mongolica.
In one embodiment of the invention, the sterilization treatment is to soak the explant with an alcohol solution and a mercuric chloride solution in sequence, and then wash the explant with sterile water. The mercuric chloride is in the form of white crystal, granule or powder, and can be used as disinfectant and antiseptic. The volume fraction of the alcohol solution may be 75%, and the mass concentration of the mercuric chloride solution may be 0.1%.
In one embodiment of the present invention, step (1) comprises: and (3) sterilizing the dormant Mongolian mulberry branches in alcohol with the mass concentration of 75% for 40-60 seconds, transferring the branches into mercuric chloride solution with the mass concentration of 0.1% for sterilization for 10-15 minutes, washing the branches with sterile water for 3-5 times, and stripping the bud tips with the length of 0.3-0.5 cm to obtain sterile explants.
The method takes the dormant mulberry branch for explant culture, and the dormant branch has the advantages that buds are not unfolded, the internal bacterium content is low, and the dormant branch is not easy to pollute. The growing branch has no dormant bud, the surface has a large amount of bacteria, and the explant infection rate is high.
(2) And after sterilization, inoculating the sterile explant into an induction culture medium for induction culture to obtain an induced seedling.
In one embodiment of the invention, the induction medium is based on MS medium and is obtained by adding 6-BA, IBA, PVP-40, sucrose and agar. The concentration of 6-BA in the induction culture medium is 1.0mg/L, the concentration of IBA is 0.5mg/L, the concentration of PVP-40 is 500mg/L, the concentration of sucrose is 20g/L, the concentration of agar is 5.5g/L, and the pH value of the induction culture medium is 5.8-6.0.
Among them, the MS culture medium is the most commonly used culture medium at present, and the formula is well known in the field of tissue culture. The culture medium has high inorganic salt concentration, and can ensure mineral nutrition required by tissue growth and accelerate the growth of callus. Because the ion concentration in the formula is high, even if some components are slightly mixed in and out in the processes of preparation, storage, disinfection and the like, the balance among ions is not influenced. The 6-BA is benzylamino adenine, is an artificially synthesized cytokinin, and has the characteristics of high efficiency, stability, low price, easy use and the like. The main function of 6-BA is to promote the formation of buds and also to induce callus formation. IBA is indolebutyric acid, which has promoting effect on the top shoot apex formation of plant twigs or buds, seedlings and the like, and can also be replaced by indoleacetic acid. PVP-40 is polyvinylpyrrolidone, and can form complex with specific polyphenol compounds (such as tannin) to improve browning problem in tissue culture. If the addition of the agent does not cause the death of the explant due to browning.
In one embodiment of the present invention, the specific conditions of the induction culture are: inoculating the sterile explant into an induction culture medium, and culturing for more than 20 days under the conditions that the temperature is 25 +/-2 ℃, the illumination intensity is 1500-2000 Lx and the illumination time is 10-14 h/day until the sterile explant grows to about 3cm, so as to obtain an induced seedling.
(3) And after the induction culture is finished, inoculating the obtained induced seedling into a subculture medium for subculture to obtain a subculture seedling.
In one embodiment of the invention, the subculture medium is based on 1/2MS medium with addition of IBA and GA3Sucrose and agar. The concentration of IBA in the subculture medium was 0.5mg/L, GA3The concentration of (3) is 3mg/L, the concentration of sucrose is 20g/L, the concentration of agar is 5.5g/L, and the pH value of the subculture medium is 5.8-6.0.
Wherein, GA3Gibberellin is used as a plant growth regulator and is mainly used for promoting the growth and development of crops and making the crops mature in advance.
The effect of subculture is: the induced seedlings need to be rapidly rejuvenated through the step so as to be proliferated and obtain a high proliferation rate. Proliferation is difficult without this step.
In one embodiment of the present invention, the specific conditions of the subculture are: inoculating the induced seedling into a subculture medium, and culturing for more than 30 days under the conditions that the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lx and the illumination time is 15-17 h/day until the induced seedling roots and grows to 5-7 cm to obtain the subculture seedling.
(4) And after the subculture is finished, inoculating the obtained subculture seedling into a multiplication culture medium for multiplication culture to obtain cluster buds.
In one embodiment of the invention, the multiplication medium is based on MS medium and is obtained by adding 6-BA, 2,4-D, sucrose and agar. The concentration of 6-BA in the multiplication medium is 1.5mg/L, the concentration of 2,4-D is 0.3mg/L, the concentration of sucrose is 30g/L, the concentration of agar is 5.5g/L, and the pH value of the multiplication medium is 5.8-6.0.
Wherein, the 2,4-D is also named as 2, 4-dichlorophenoxyacetic acid, which is one of plant growth regulators. Can induce the formation of woody plant callus.
In one embodiment of the present invention, the specific conditions of the propagation culture are: and cutting a small section with an axillary bud from the subculture seedling, inoculating the small section into a proliferation culture medium, and culturing for more than 30 days under the conditions that the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lx and the illumination time is 15-17 h/day until the small section of the subculture seedling grows to 5-7 cm, so as to obtain the cluster bud.
(5) And after the propagation culture is finished, inoculating the obtained cluster buds into a rooting culture medium for rooting culture to obtain rooted seedlings.
In one embodiment of the invention, the rooting medium is obtained by taking 1/2MS medium as a basis and adding IBA, sucrose and agar, wherein the concentration of IBA in the rooting medium is 0.3mg/L, the concentration of sucrose is 20g/L, the concentration of agar is 5.5g/L, and the pH value of the rooting medium is 5.8-6.0.
In one embodiment of the present invention, the specific conditions for rooting culture are: dividing the cluster buds into single plants, inoculating the single plants into a rooting culture medium, and performing rooting culture for 20-25 days under the conditions that the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lx and the illumination time is 15-17 h/d to obtain rooted seedlings.
Further, after the step (5) is finished, the method further comprises the step (6): and (4) cleaning the rooting seedlings, transferring the rooting seedlings to a matrix for culture, and culturing the rooting seedlings in the matrix for 15-20 days to obtain the transplanted seedlings. During the period, the environmental humidity is kept between 80 and 90 percent, and the day and night temperature is between 20 and 25 ℃. And gradually reducing the humidity after transplanting for 1 week, and hardening the seedlings for 15-20 days in total to obtain the transplanted seedlings.
In one embodiment of the invention, the volume ratio of the turf, the vermiculite and the perlite in the matrix is 3: 2: 1. the vermiculite is a natural, inorganic and nontoxic mineral substance, has good water permeability, and can effectively promote the growth of plant roots and the stable development of seedlings. Provides water and nutrition necessary for plant growth for a long time, and can keep the temperature of the sunlight of the roots stable. The vermiculite can make crops obtain sufficient water and mineral substances from the early growth stage, promote the plants to grow faster and increase the yield. As the transplanted rooted seedlings grow at a high speed, the matrix needs to contain more nutrients, and turf is added into the matrix. The perlite is a white granular material with a cellular structure inside, which is prepared by preheating, instantaneous high-temperature roasting and expanding perlite ore sand and has a porous structure. The porosity of the product greatly promotes the growth and development of the fibrous root system of the plant and has excellent fixing effect on the tree.
The method provided by the invention effectively solves the problems of variation and slow growth generated by the reproduction of the Mongolian mulberry seeds and the spread of plant diseases and insect pests generated by a cuttage mode. The method can make the proliferation stage of Mongolian mulberry grow at 9.3 times of rate, and the rooting rate is 100%.
Examples 1 to 1
(1) And (3) lightly brushing the dormant mulberry branches with washing powder water with the mass concentration of 0.5%, and washing with tap water until no foam exists. In an ultra-clean workbench, the branches with dormant buds are soaked in 75% alcohol by volume for 40s, then washed with sterile water for 3-5 times, then disinfected with 0.1% mercuric chloride solution by mass concentration for 15min, and then washed with sterile water for 5 times.
(2) And (3) stripping the bud tip with the length of 0.3-0.5 cm from the branch sterilized in the step (1) by using a scalpel, and inoculating the bud tip into an induction culture medium. The induction culture medium is obtained by taking an MS culture medium as a base and adding 6-BA, IBA, PVP-40, sucrose and agar, wherein the concentration of the 6-BA in the induction culture medium is 1.0mg/L, the concentration of the IBA is 0.5mg/L, the concentration of the PVP-40 is 500mg/L, the concentration of the sucrose is 20g/L, the concentration of the agar is 5.5g/L, and the pH value is 5.8-6.0. Culturing at 25 +/-2 ℃, with illumination intensity of 1500-2000 lx and illumination time of 12h/d, obtaining induced seedlings when the seedlings grow to about 3cm after 20 days of culture, and then carrying out subculture;
(3) and (3) inoculating the induced seedling obtained in the step (2) into a subculture medium. The subculture medium is based on 1/2MS medium, and IBA and GA are added3Sucrose and agar, the concentration of IBA in the subculture medium is 0.5mg/L, GA3The concentration of (A) is 3mg/L, the concentration of sucrose is 20g/L, the concentration of agar is 5.5g/L, and the pH value is 5.8-6.0. Culturing at the temperature of 25 +/-2 ℃, with the illumination intensity of 2500-3000 lx and the illumination time of 16h/d, culturing for 30 days to obtain a subculture seedling when the seedling grows to 5-7 cm, and then performing proliferation culture;
(4) and (4) selecting a part with low lignification degree from the secondary seedlings obtained in the step (3), and cutting a small section with an axillary bud. This part has a low lignification degree and is liable to generate clumpy buds. The pellet was inoculated into proliferation medium. The multiplication culture medium is obtained by taking an MS culture medium as a base and adding 6-BA, 2,4-D, sucrose and agar, wherein the concentration of the 6-BA is 1.5mg/L, the concentration of the 2,4-D is 0.3mg/L, the concentration of the sucrose is 30g/L, the concentration of the agar is 5.5g/L, and the pH value is 5.8-6.0. Culturing at 25 +/-2 ℃, with illumination intensity of 2500-3000 Lx and illumination time of 16h/d for 30 days, then growing the small segment of subculture seedlings to 5-7 cm to obtain cluster buds, and then carrying out rooting culture.
(5) Dividing the cluster buds obtained in the step (4) into single plants, and inoculating the single plants in a rooting culture medium. The rooting medium is obtained by taking 1/2MS medium as a basis and adding IBA, sucrose and agar, wherein the concentration of the IBA is 0.3mg/L, the concentration of the sucrose is 20g/L, the concentration of the agar is 5.5g/L, and the pH value is 5.8-6.0. And (3) carrying out rooting culture for 20 days under the conditions that the culture temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lx and the illumination time is 16h/d to obtain the rooted seedlings.
(6) Placing the rooted seedlings obtained in the step (5) on a greenhouse seedling bed 2 weeks before bottle discharging, keeping the environmental temperature at 25 +/-2 ℃, and performing transition exercise with the illumination intensity of 1500-2000 lx. The volume ratio of grass carbon to vermiculite to perlite is 3 to 2 to 1. Washing residual culture medium with normal temperature clear water, implanting the cleaned seedling into a plug tray, and spraying once with normal temperature clear water. During the period, the environmental humidity is kept between 80% and 90%, and the day and night temperature is between 20 ℃ and 25 ℃. After 1 week, the humidity was gradually decreased. After 15 days, hardening off the seedlings, and planting.
Examples 1-2 to examples 1-5
And (3) changing the types and the dosage of the reagents in the induction culture medium in the step (2), and observing the influence of different hormone combinations on the germination rate and the growth condition of the Mongolian mulberry. The amounts of sucrose and agar used, and the culturing method were the same as in example 1-1, and the results are shown in Table 1.
TABLE 1
As can be seen from Table 1, the average plant height and growth of the Mongolian-induced seedlings were reduced by varying the concentrations of 6-BA, IBA and PVP-40, as compared to example 1-1. Thus for the induction culture step, the most suitable induction medium formulation is MS medium +1.0mg/L of 6-BA +0.5mg/L of IBA +500mg/L of PVP-40.
Examples 1-6 to examples 1-9
And (4) changing the types and the dosage of the reagents in the subculture medium in the step (3), and observing the influence of different hormone combinations on the growth of the Mongolian mulberry. The amounts of sucrose and agar used, and the culturing method were the same as in example 1-1, and the results are shown in Table 2. The results were calculated by averaging.
TABLE 2
As can be seen from Table 2, IBA and GA were changed as compared with example 1-13The average plant height and the number of stem nodes of the Mongolian mulberry subculture seedlings are reduced. Therefore, for the subculture step, the optimum subculture medium formulation is 1/2MS medium +0.5mg/L IBA +3mg/L GA3。
Examples 1-10 to examples 1-13
And (4) changing the types and the dosage of the reagents in the proliferation culture medium in the step (4), and observing the influence of different hormone combinations on the growth of the Mongolian mulberry. The amounts of sucrose and agar used, and the culturing method were the same as in example 1-1, and the results are shown in Table 3. The results were calculated by averaging.
TABLE 3
As can be seen from Table 3, the proliferation rate and the average plant height of the tufted buds of Morus mongolica were decreased by changing the addition concentrations of 6-BA and 2,4-D as compared with example 1-1. Therefore, for the multiplication culture step, the most suitable formula of the multiplication medium is MS medium +1.5mg/L of 6-BA +0.3mg/L of 2, 4-D.
Examples 1-14 to examples 1-15
The light conditions in the rooting culture process of step (5) were changed, and the other culture methods were the same as in example 1-1. The influence on the rooting rate and the number of roots of the Mongolian mulberry is observed, and the result is shown in table 4.
TABLE 4
As can be seen from Table 4, the rooting rate of the Mongolian mulberry rooted seedlings is reduced and the growth conditions are obviously different by changing the illumination intensity compared with the example 1-1. Therefore, the optimum illumination intensity for the rooting culture step is 2500-3000 Lx.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims (3)

1. A tissue culture method of Mongolian mulberry seedlings is characterized by comprising the following steps:
(1) taking a Mongolian mulberry explant, and carrying out sterilization treatment to obtain a sterile explant, wherein the explant is a dormant bud of the Mongolian mulberry;
(2) inoculating the sterile explant into an induction culture medium for induction culture to obtain an induced seedling;
the induction culture medium is obtained by taking an MS culture medium as a basis and adding 6-BA, IBA, PVP-40, cane sugar and agar, wherein the concentration of the 6-BA in the induction culture medium is 1.0mg/L, the concentration of the IBA is 0.5mg/L, the concentration of the PVP-40 is 500mg/L, the concentration of the cane sugar is 20g/L, the concentration of the agar is 5.5g/L, and the pH value of the induction culture medium is 5.8-6.0;
the induction culture is to inoculate the sterile explant into an induction culture medium, and culture the sterile explant for more than 20 days under the conditions that the temperature is 25 +/-2 ℃, the illumination intensity is 1500-2000 Lx and the illumination time is 10-14 h/day until the sterile explant grows to about 3cm, so as to obtain an induced seedling;
(3) inoculating the induced seedling into a subculture medium for subculture to obtain a subculture seedling;
the subculture medium is based on 1/2MS culture medium, and IBA and GA are added3Sucrose and agar, the concentration of IBA in the subculture medium is 0.5mg/L, and GA3The concentration of the culture medium is 3mg/L, the concentration of the sucrose is 20g/L, the concentration of the agar is 5.5g/L, and the pH value of the subculture medium is 5.8-6.0;
the subculture is to inoculate the induced seedling into a subculture medium, and culture the induced seedling for more than 30 days under the conditions that the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lx and the illumination time is 15-17 h/day until the induced seedling roots and grows to 5-7 cm, so as to obtain the subculture seedling;
(4) inoculating the subculture seedling into a proliferation culture medium for proliferation culture to obtain cluster buds;
the enrichment culture medium is obtained by taking an MS culture medium as a base and adding 6-BA, 2,4-D, sucrose and agar, wherein the concentration of the 6-BA in the enrichment culture medium is 1.5mg/L, the concentration of the 2,4-D in the enrichment culture medium is 0.3mg/L, the concentration of the sucrose in the enrichment culture medium is 30g/L, the concentration of the agar in the enrichment culture medium is 5.5g/L, and the pH value of the enrichment culture medium is 5.8-6.0;
the propagation culture is to cut a small section with an axillary bud from the subculture seedling, inoculate the small section in a propagation culture medium, and culture for more than 30 days under the conditions that the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lx, and the illumination time is 15-17 h/day until the small section of the subculture seedling grows to 5-7 cm, so as to obtain cluster buds;
(5) inoculating the cluster buds into a rooting culture medium for rooting culture to obtain rooted seedlings;
the rooting medium is obtained by taking 1/2MS medium as a basis and adding IBA, sucrose and agar, wherein the concentration of IBA in the rooting medium is 0.3mg/L, the concentration of sucrose is 20g/L, the concentration of agar is 5.5g/L, and the pH value of the rooting medium is 5.8-6.0;
and the rooting culture is to inoculate the cluster buds into a rooting culture medium, and carry out rooting culture for 20-25 days under the conditions that the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lx and the illumination time is 15-17 h/d, so as to obtain rooted seedlings.
2. The method according to claim 1, wherein in the step (1), the sterilization treatment is that the explant is soaked by using an alcohol solution and a mercuric chloride solution in sequence and then washed by using sterile water.
3. The method of claim 1, wherein after the step (5) is finished, the method further comprises the step (6): and (4) cleaning the rooted seedlings, and culturing in a matrix for 15-20 days to obtain transplanted seedlings.
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